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Development of an Ecotoxicological Test with a Soil Microalgae Species Development of an Ecotoxicological Test with a Soil Microalgae Species Vânia Gonçalves Carvalhido Master’s degree in Biology and Water Management Biology Department 2018 Supervisor Ruth Maria de Oliveira Pereira, Assistant Professor with Habilitation, Faculty of Sciences, University of Porto Co-supervisor Paula Maria Vieira de Melo Gomes, Assistant Professor, Faculty of Sciences, University of Porto Todas as correções determinadas pelo júri, e só essas, foram efetuadas. O Presidente do Júri, Porto, ______/______/__ _______ Vânia Gonçalves Carvalhido Development of an Ecotoxicological Test with a Soil Microalgae Species Dissertação de Mestrado em Biologia e Gestão da Qualidade da Água apresentada à Faculdade de Ciências da Universidade do Porto Departamento de Biologia Faculdade de Ciências da Universidade do Porto 2018 Development of an Ecotoxicological Test with a Soil Microalgae Species Acknowledgements First, I would like to express my enormous gratitude to my parents for always being there for me and for their immense and exhausting sacrifice through all of these years. For all the love, advice and for just being there for me in my moments of crisis. To both of you, thank you so much. I would like to express my gratitude to both of my supervisors, Professor Ruth Pereira and Professor Paula Melo for everything I learn this year, for helping me with the execution of my experiments, for their patience, care, and support through this year. Both were an important support in moments of doubt, making me less stressed, for all the advice and important guidance. I would like to also thank Professor Paula Tamagnini and Ângela Brito for all the help and knowledge with the molecular techniques. I also want to express my gratitude to Diogo Coelho for all the help in the identification of my species, for helping me in the laboratory and for all the conversations throughout the year. I would like to thank everyone involved in my dissertation, with a special thank you to Ana Ribeiro for helping me with planning and the execution of my experiments, to Inês Nogueira for helping me with the statistical program and to everyone else for being helpful every time I asked for a favor. To my parents and my grandmother, I dedicate this dissertation, for their presence in my life and for making me a better human being. Last but not least, I would like to thank my brother and to my dog for keeping me company and listening to my frustrations and to Ana Rocha, for always being there for a kind word and support, even at distance. I Development of an Ecotoxicological Test with a Soil Microalgae Species Contents Index Acknowledgements ..................................................................................................................................... I Contents Index ............................................................................................................................................ II Figures Index ............................................................................................................................................. IV Tables Index ............................................................................................................................................... VI Abstract ...................................................................................................................................................... VII Resumo ....................................................................................................................................................... IX 1. Introduction ......................................................................................................................................... 1 1.1. Terrestrial microalgae ............................................................................................................... 1 1.2. Terrestrial Ecotoxicology .......................................................................................................... 4 2. Aim of the thesis............................................................................................................................... 12 3. Material and Methods.......................................................................................................................... 13 3.1. Microalgae isolation and culture conditions ............................................................................. 13 3.2. Genetic Identification of the BSC selected microalgae ......................................................... 14 3.2.1. DNA extraction ................................................................................................................ 14 3.2.2. PCR amplification ........................................................................................................... 14 3.2.3. DNA purification and sequencing ................................................................................. 15 3.3. Ecotoxicological tests in aqueous medium following available standard protocol for microalgae ............................................................................................................................................. 16 3.3.1. BSC microalga growth curves in a liquid medium ..................................................... 16 3.3.2. Reference substances test solutions ........................................................................... 17 3.3.3. Ecotoxicological test in BG11 medium with the BSC microalga ............................. 17 3.4.1. Standard artificial soil ........................................................................................................... 19 3.4.2. BSC microalga growth curves in soil................................................................................. 20 3.4.3. Pigment content .................................................................................................................... 21 3.4.4. Preparation of test solutions ............................................................................................... 22 3.4.5. Ecotoxicological test in the OECD soil and with the BSC microalga ........................... 22 3.5. Statistical analysis.................................................................................................................... 23 II Development of an Ecotoxicological Test with a Soil Microalgae Species 4. Results ............................................................................................................................................... 24 4.1. Identification of the BSC selected microalga ........................................................................... 24 4.1.2. Genetic identification of the BSC selected microalga .............................................. 24 4.2. Ecotoxicological test in liquid medium with the BSC microalga ...................................... 26 4.2.1. Growth curves and ecotoxicological tests in aqueous medium following the available standard protocol for freshwater microalgae and cyanobacteria ........................... 26 4.2.2. Ecotoxicological test in BG11 medium with M. inermum ......................................... 27 4.3. Ecotoxicological test in artificial soil with the BSC microalga .......................................... 29 4.3.1. M. inermum growth curve in artificial OECD soil ....................................................... 29 4.3.2. Ecotoxicological test with M. inermum in OECD artificial soil ................................. 30 5. Discussion ......................................................................................................................................... 33 5.1. Characterization and identification of a selected microalga from the BSC .................... 33 5.2. Ecotoxicological Assays in Aqueous Medium Following Available Standard Protocol for Freshwater Microalgae and Cyanobacteria..................................................................................... 35 M. inermum ....................................................................................................................................... 37 Chlorella vulgaris ............................................................................................................................. 37 M. inermum ....................................................................................................................................... 39 Scenedesmus obliquuos ................................................................................................................. 39 5.3. Ecotoxicological Test with Soil BSC Microalgae using Artificial OECD Soil as a Test Substance.............................................................................................................................................. 40 6. Conclusions ...................................................................................................................................... 43 References ................................................................................................................................................ 44 Annex A ...................................................................................................................................................... 69 Annex B ...................................................................................................................................................... 71 III Development of an Ecotoxicological Test with a Soil Microalgae Species Figures Index Figure 1 - Inoculum
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